Local Structure in Spider Dragline Silk Investigated by Two-Dimensional Spin-Diffusion Nuclear Magnetic Resonance

Abstract

The local structure of dragline silk from the spider Nephila madagascariensis is investigated by solid-state nuclear magnetic resonance. Two-dimensional (2D) spin-diffusion experiments show that the alanine-rich domains of the protein form /9-sheet structures in agreement with one-dimensional NMR results from a different species of the genus Nephila (Simons, A.; Ray, E.; Jelinski, L. W. Macromolecules 1994, 27, 5235) but at variance with diffraction results. The micro structure of the glycine-rich domains is found to be ordered, The simplest model that explains the experimental findings is a 3rhelical structure. Random coils, planar /3-sheets, and a-helical conformations are not found in significant amounts in the glycine-rich domains. This observation may help to explain the extraordinary mechanical properties of this silk, because 3\-helices can form interhelix hydrogen bonds.( 1) In t r o d u c t io nSpider dragline silk is a rem ark ab le biopolymer: its mechanical properties are a u n iq u e combination of high tensile strength and high elasticity.1" 4 In contrast to the synthesis of man-made high-perform ance m aterials (e.g. steel or Kevlar) spider silk is synthesized a t am bient tem perature and pressure. The polypeptides th a t form the spider's dragline are produced in a set of glands (the m ajor ampullate) a n d channeled through a duct to the spigot.6»6 It seems th a t th e solvent (water) is extracted in the duct, and th a t th e silk goes into a liquid crystalline phase.7,8 A t th e end of the duct the silk is drawn through a valve w here it apparently forms the fiber, now insoluble in all b u t th e m ost aggressive solvents; remaining free w ater evaporates quickly in the a ir.9 The extraordinary properties of th e spider's drag line silk make it attractive to th in k about technically produced analogues w ith well-defined amino-acid se quences (see ref 1 0 and references therein). To do so, it is essential to understand th e macroscopic properties of dragline silk in term s of its microscopic structure including the amino-acid sequence of the protein (the prim ary structure), the (local) folding of the strands (its secondary structure), the packing arrangem ent (crystal line or amorphous regions) in the solid fiber, and possible superstructures. The observations th a t w ater can act as a plasticizer for som e of th e silks produced by spiders11" 13 and th a t w etting of the dragline silk can lead to a phenomenon called su p er contraction9 are a clear indication th a t the knowledge of the amino-acid sequence alone is not sufficient to u n d erstan d th e properties of silk but th a t detailed knowledge of the secondary structure, the packing, and th e interplay with resident and am bient w ater is required.Solid-state NMR can probe th e local stru ctu re in disordered systems where the lack of translational longrange order makes the application of diffraction techt Presented in part at the 36th Experimental NMR conference,

Molecular and Nanostructural Mechanisms of Deformation, Strength and Toughness of Spider Silk Fibrils

“…At length-scales on the order of hundreds of nanometers, spider silk appears as an entanglement of polypeptide chains, with two distinct domains that consist of (1) a semi-amorphous region [8,33] and (2) a highly-ordered crystalline domain [22]. We model the silk constitutive unit as a serial arrangement of these two domains, namely a crystalline beta-sheet region and a semi-amorphous domain as shown in Fig.…”

“…1a) [6][7][8]. Thereby, the anti-parallel beta-sheet nanocrystals [9][10][11][12] play a key role in defining the mechanical properties as they provide stiff orderly cross-linking domains embedded in a semiamorphous matrix that consists of less orderly beta-structures, 3 1 helices and beta-turns [6,13,14].…”

Structure of silk by raman spectromicroscopy: From the spinning glands to the fibers

“…11,[44][45][46][47] Comparison between the b-sheet content determined from the spectroscopic data and the percentage of amino acids that may potentially be included into the b-sheets lead to the conclusion that for the dragline fiber the AG and GGA motifs adjacent to the (A) n are incorporated into the b-sheets, 33 which is consistent with the literature. [48][49][50] Other results have shown that the minor ampullate (Mi) and cylindriform (Cyl) silks belong to the same structural family than the Ma silk as they are composed of highly oriented b-sheets (35-37%) dispersed in an amorphous matrix made of other slightly oriented secondary structures. Raman spectra have also revealed in the Mi and Cyl fibers the presence of some disordered conformations that are reminiscent of the native conformations and that are present in the dope but not in the Ma fiber.…”

Orientational order of Australian spider silks as determined by solid‐state NMR

“…13 However, debate on the structure of the poly-Gly regions continues. Once thought to be entirely amorphous, the structure of the Gly-rich region may be semiordered in a 3 1 helix [14][15][16] or Type 1 turn. 11,17 Silk from the silkworm Bombyx mori has been extensively studied and is a fibrous protein consisting of a repeating sequence of (GAGAGS) somewhat similar in composition to spider silk and contains Gly (43.7%), Ala (28.8%), and Ser (11.9%).…”

“…Evidence from Rotational Echo Double Resonance NMR experiments 17 suggests that this third phase forms compact turn-like structures. Two-dimensional (2D) spin diffusion NMR has demonstrated that the Gly-rich regions are ordered and form 3 1 -helices, 14 but these experiments, however, do not distinguish between ordered Gly-rich regions from Gly-rich amorphous regions. 11 While most NMR results conclude that the Ala-rich regions form -sheet structures, Jelinski and coworkers, 1,10 using carbon and deuterium NMR relaxation times of N. clavipes dragline silk, further described Ala as present in two distinct motional environments, one highly oriented (40%) and another poorly oriented (60%).…”